It is clear that inactivation of PTEN is a major contributor to the evolution of hormone-refractory prostate cancer. The function of PTEN as a tumor suppressor is most closely linked to its activity as a lipid phosphatase (Maehama and Dixon, 1998) and hence its ability to attenuate signaling through the phosphoinositide 3-kinase pathway. In the initial funding period our work focused on the role of PTEN as a cell-cycle regulator. Here,we showed that the PTEN lipid phosphatase activity and regulation of Akt was essential for PTEN to arrest cells in G1 (Ramaswamy et al., 1999). Elegant genetic studies placing PTEN in the dauer pathway in C.elegans led us to ask whether human FOXO homologues of daf-16, might play a role in PTEN-mediate tumor suppression. Indeed, we found that Foxol is constitutively inactivated in PTEN null cells and its reconstitution is sufficient to reverse the transformed phenotype of PTEN-null cancer cell lines (Nakamura et al., 2000). Surprisingly, we found that this Foxol activity did not require binding to an insulin-response element and instead was associated with the ability of FOX01 to inhibit cyclin D1 and D2 transcription (Ramaswamy et al., 2002). Preliminary data strongly suggest that FOX01 can act as a transcriptional repressor through interactions with the proto-oncogene SKI and that this activity is linked to its ability to induce a cell-cycle arrest and suppress tumor growth. Based on this data we propose in specific Aim 1:1. To test the hypothesis that a repressor complex mediates the cell-cycle regulatory properties of FOXO1- During the original grant period we were the first group to demonstrate that PTEN is phosphorylated on residues within the C-terminal 'tail' and that these phosphorylation events appear to regulate PTEN stability. Moreover, we showed that the unphosphorylated form of PTEN is found in a more open conformation, associates with a high molecular weight complex, and is more active in biological assays. Based on this data we proposed a model whereby the majority of cellular PTEN is phosphorylated and in a relatively inactive state, upon dephosphorylation PTEN can then enter a so-called PTEN-associated complex (PAC) (Vazquez et al., 2001). In this context PTEN is more active yet subjected to degradation. Data from other labs have been entirely consistent with this model, yet an understanding of the mechanism through which phosphorylation regulates PTEN, the localization of the unphosphorylated forms of PTEN, and the nature of the PAC remain obscure. Based on this data we propose in specific aims 2 and 3: 2. To generate antibody reagents that selectively recognizes unphosphorylated PTEN. 3. To purify and identify proteins in the PTEN associated complex (PAC).